Heterodimeric polypeptides have been designed by engineering the CH3 domain in the Fc region of an antibody (for instance: Ridgway et. al. Protein Engineering 1996, 9, 617-621; U.S. Pat. No. 5,731,168; U.S. Pat. No. 5,821,333; U.S. Pat. No. 5,807,706; U.S. Pat. No. 7,183,076; U.S. Pat. No. 7,642,228; U.S. Pat. No. 7,695,936; US20100286374; US20070287170 and US20120149876). This method has been used to design bi-specific antibodies, and express non-glycosylated forms of such antibodies from non-mammalian cell lines such as E. coli (see, for instance, Merchant et. al. Nat. Biotechnol. 1998; 16; 677-681, or Jin et al. Cancer Res 2008; 68; 4360-4368). Typically, screening for a stable cell culture expressing the pure or homogeneous product molecule in the heterodimeric form without contaminant homodimers is challenging. The heterodimeric product of interest is co-expressed in the cell with contaminant homodimers and monomers and it is challenging to obtain the pure heterodimeric species (see for example, US Patent Publication No. 20090232811).
Large scale expression of these engineered Fe heterodimers and bi-specific antibodies comprising the same in mammalian cell lines, has been hindered by the unreliable and variable purity of the heterodimer obtained (see for instance Jackman et. al. J. Bioi. Chern. 2010, 285, 20850-20859). Among other approaches, currently, an E. coli based expression strategy is used for large-scale expression. Alternately, two heavy chains of the asymmetric antibody are expressed as separate heavy-light chain fragments in two different cell lines. The two purified fragments are then annealed in a one-to-one ratio. A similar procedure for producing asymmetric antibodies by mixing two different antibodies is utilized by Schuurman et. al. (US201 001 05874A1). This is a multistep process and leads to high cost of goods.
Some methods of co-expression of the heavy chains of a symmetric antibody in E. coli have been developed (for instance Cabilly et. al: U.S. Pat. No. 4,816,567; U.S. Pat. No. 6,331,415; U.S. Pat. No. 7,923,221). However, these methods are not developed for expression of asymmetric antibodies, and the process of expression in E. coli results in an aglycosylated antibody Fc which has characteristics that are very different from the intrinsic characteristics of the wild type antibody which is glycosylated. Accordingly, the large scale production of asymmetric antibody has been limited and it has been difficult to achieve high purity and yield in stable mammalian cell lines.